This research is concerned with the dynamic behavior of intracellular Ca2+ in hippocampal neurons. In neurons Ca2+ acts as a second messenger coupling synaptic. activity and action potential generation to multiple intracellular processes. While intracellular mechanisms may shape the spatial and temporal patterns of Ca + signals and thus modulate Ca2+- dependent events, the fate of Ca2+ ions after they enter the cytoplasm is less well understood than the mechanisms by which they cross the cell membrane. We have studied intracellular mechanisms of Ca2+ release in cultured mouse hippocampal neurons. We localized Ca2+ release channels of endoplasmic reticulum in somas and dendrites using specific antibodies and confocal microscopy, and concluded that ryanodine receptor channels (mediating Ca2+-induced Ca2+ release) and inositol 1,4,5-trisphosphate (InsP3-) receptor channels (mediating InsP3-gated Ca2+ release) are not homogeneously distributed. We also used Ca2+-sensitive dyes to image and measure cytoplasmic Ca2+ concentrations ([Ca2+]) while inducing activation of ryanodine receptors (with caffeine) and InsP3 receptors (with muscarinic acetylcholine receptor agonists). We observed that acetylcholine induced [Ca2+] fluctuations in small dendritic regions, while caffeine induced more widely distributed and sustained increases in [Ca2+]. Our three Specific Aims further study of Ca2+ release in hippocampal neurons. 1. To examine the intracellular distributions of InsP3 and ryanodine receptors in comparison to the distributions of endoplasmic reticulum and cytoskeletal elements. These experiments will test hypotheses that Ca2+ release channels may be linked to the cytoskeleton and that.disruption of microtubules and/or microfilaments will distort or inhibit intracellular Ca2+ release. 2. To examine fluctuations in dendritic Ca2+ to determine if the distribute randomly or propagate in an orderly manner. We will image larger portions of dendritic fields at higher temporal resolutions than i our previous experiments to examine regions of elevated (Ca2+] after stimulation with caffeine or acetylcholine and after local dendritic activation with iontophoretically applied glutamate or acetylcholine, or synaptic activation. We will test the hypothesis that the dendritic cytoplasm may support propagating Ca2+ waves. 3. To examine dendrites for restricted domains of high (micromolar) Ca2+ levels associated with aggregations of ryanodine or InsP3 receptors. We will image the distribution of [Ca2+] during stimulation using fluorescent dyes with low Ca2+ affinity, and compare regions of high [Ca2+] to the locations of Ca2+ release channels. If they are in registration, these observations would suggest that one function of intracellular Ca2+ release in neurons is to effect local activation of Ca2+-dependent processes.